Interleukin 1 ("IL-1") is a major pro-inflammatory and immunoregulatory protein that stimulates fibroblast differentiation and proliferation, the production of prostaglandins, collagenase and phospholipase by synovial cells and chondrocytes, basophil and eosinophil degranulation and neutrophil activation. Oppenheim, J. H. et al., Immunology Today, 7, pp. 45-56 (1986). As such, it is involved in the pathogenesis of chronic and acute inflammatory and autoimmune diseases. For example, in rheumatoid arthritis, IL-1 is both a mediator of inflammatory symptoms and of the destruction of the cartilage proteoglycan in afflicted joints. Wood, D. D. et al., Arthritis Rheum. 26, p. 975, (1983); Pettipher, E. J. et al., Proc. Natl. Acad. Sci. U.S.A. 71, p. 295 (1986); Arend, W. P. and Dayer, J. M., Arthritis Rheum. 38, p. 151 (1995). IL-1 is also a highly potent bone resorption agent. Jandiski, J. J., J. Oral Path 17, p. 145 (1988); Dewhirst, F. E. et al., J. Immunol. 8, p. 2562 1985). It is alternately referred to as "osteoclast activating factor" in destructive bone diseases such as osteoarthritis and multiple myeloma. Bataille, R. et al., Int. J. Clin. Lab. Res. 21(4), p. 283 (1992). In certain proliferative disorders, such as acute myelogenous leukemia and multiple myeloma, IL-1 can promote tumor cell growth and adhesion. Bani, M. R., J. Natl. Cancer Inst. 83, p. 123 (1991); Vidal-Vanaclocha, F., Cancer Res. 54, p. 2667 (1994). In these disorders, IL-1 also stimulates production of other cytokines such as IL-6, which can modulate tumor development (Tartour et al., Cancer Res. 54, p. 6243 (1994). IL-1 is predominantly produced by peripheral blood monocytes as part of the inflammatory response and exists in two distinct agonist forms, IL-1.alpha. and IL-1.beta.. Mosely, B. S. et al., Proc. Nat. Acad. Sci., 84, pp.4572-4576 (1987); Lonnemann, G. et al., Eur.J. Immunol., 19, pp.1531-1536 (1989).
IL-1.beta. is synthesized as a biologically inactive precursor, pIL-1.beta.. pIL-1.beta. lacks a conventional leader sequence and is not processed by a signal peptidase. March, C. J., Nature, 315, pp.641-647 (1985). Instead, pIL-1.beta. is cleaved by interleukin-1.beta. converting enzyme ("ICE") between Asp-116 and Ala-117 to produce the biologically active C-terminal fragment found in human serum and synovial fluid. Sleath, P. R. et al., J. Biol. Chem., 265, pp.14526-14528 (1992); Howard, A. D. et al., J. Immunol., 147, pp.2964-2969 (1991). ICE is a cysteine protease localized primarily in monocytes. It converts precursor IL-1.beta. to the mature form. Black, R. A. et al., FEBS Lett., 247, pp.386-390 (1989); Kostura, M. J. et al., Proc. Natl. Acad. Sci. U.S.A., 86, pp.5227-5231 (1989). Processing by ICE is also necessary for the transport of mature IL-1.beta. through the cell membrane.
ICE, or its homologs, also appears to be involved in the regulation of programmed cell death or apoptosis. Yuan, J. et al., Cell, 75, pp.641-652 (1993); Miura, M. et al., Cell, 75, pp. 653-660 (1993); Nett-Fiordalisi, M. A. et al., J. Cell Biochem., 17B, p.117 (1993). In particular, ICE or ICE homologs are thought to be associated with the regulation of apoptosis in neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. Marx, J. and Baringa, M. Science, 259, pp.760-762 (1993); Gagliardini, V. et al., Science, 263, pp.826-828 (1994). Therapeutic applications for inhibition of apoptosis may include treatment of Alzheimer's disease, Parkinson's disease, stroke, myocardial infarction, spinal atrophy, and aging.
ICE has been demonstrated to mediate apoptosis (programmed cell death) in certain tissue types. Steller, H., Science, 267, p. 1445 (1995); Whyte, M. and Evan, G., Nature, 376, p. 17 (1995); Martin, S. J. and Green, D. R., Cell, 82, p. 349 (1995); Alnemri, E. S., et al., J. Biol. Chem., 270, p. 4312 (1995); Yuan, J. Curr. Opin. Cell Biol., 7, p. 211 (1995). A transgenic mouse with a disruption of the ICE gene is deficient in Fas-mediated apoptosis (Kuida, K. et al., Science 267, p. 2000 (1995)). This activity of ICE is distinct from its role as the processing enzyme for pro-IL1.beta.. It is conceivable that in certain tissue types, inhibition of ICE may not affect secretion of mature IL-1.beta., but may inhibit apoptosis.
Enzymatically active ICE has been previously described as a heterodimer composed of two subunits, p20 and p10 (20 kDa and 10 kDa molecular weight, respectively). These subunits are derived from a 45 kDa proenzyme (p45) by way of a p30 form, through an activation mechanism that is autocatalytic. Thornberry, N. A. et al., Nature, 356, pp.768-774 (1992). The ICE proenzyme has been divided into several functional domains: a prodomain (p14), a p22/20 subunit, a polypeptide linker and a p10 subunit. Thornberry et al., supra; Casano et al., Genomics, 20, pp. 474-481 (1994).
Full length p45 has been characterized by its cDNA and amino acid sequences. PCT patent applications WO 91/15577 and WO 94/00154. The p20 and p10 cDNA and amino acid sequences are also known. Thornberry et al., supra. Murine and rat ICE have also been sequenced and cloned. They have high amino acid and nucleic acid sequence homology to human ICE. Miller, D. K. et al., Ann. N.Y. Acad. Sci., 696, pp. 133-148 (1993); Molineaux, S. M. et al., Proc. Nat. Acad. Sci., 90, pp. 1809-1813 (1993). The three-dimensional structure of ICE has been determined at atomic resolution by X-ray crystallography. Wilson, K. P. et al., Nature, 370, pp. 270-275 (1994). The active enzyme exists as a tetramer of two p20 and two p10 subunits.
Additionally, there exist human homologs of ICE with sequence similarities in the active site regions of the enzymes. Such homologs include TX (or ICE.sub.rel-II or ICH-2) (Faucheu, et al., EMBO J., 14, p. 1914 (1995); Kamens J., et al., J. Biol. Chem., 270, p. 15250 (1995); Nicholson et al., J. Biol. Chem., 270 p. 15870 (1995)), TY (or ICE.sub.rel-III) (Nicholson et al., J. Biol. Chem., 270, p. 15870 (1995); ICH-1 (or Nedd-2) (Wang, L. et al., Cell, 78, p. 739 (1994)), MCH-2, (Fernandes-Alnemri, T. et al., Cancer Res., 55, p. 2737 (1995), CPP32 (or YAMA or apopain) (Fernandes-Alnemri, T. et al., J. Biol. Chem., 269, p. 30761 (1994); Nicholson, D. W. et al., Nature, 376, p. 37 (1995)), and CMH-1 (or MCH-3) (Lippke et al., J. Biol. Chem., (1996); Fernandes-Alnemri, T. et al., Cancer Res., (1995)). Each of these ICE homologs, as well as ICE itself, is capable of inducing apoptosis when overexpressed in transfected cell lines. Inhibition of one or more of these homologs with the peptidyl ICE inhibitor Tyr-Val-Ala-Asp-chloromethylketone results in inhibition of apoptosis in primary cells or cell lines. Lazebnik et al., Nature, 371, p. 346 (1994). The compounds described herein are also capable of inhibiting one or more homologs of ICE. Therefore, these compounds may be used to inhibit apoptosis in tissue types that contain ICE homologs.
Interferon-gamma inducing factor (IGIF) is an approximately 18-kDa polypeptide that stimulates T-cell production of interferon-gamma (IFN-.gamma.). IGIF is produced by activated Kupffer cells and macrophages in vivo and is exported out of such cells upon endotoxin stimulation. Thus, a compound that decreases IGIF production would be useful as an inhibitor of such T-cell stimulation which in turn would reduce the levels of IFN-.gamma. production by those cells.
IFN-.gamma. is a cytokine with immunomodulatory effects on a variety of immune cells. In particular, IFN-.gamma. is involved in macrophage activation and Th1 cell selection (Belardelli, F. APMIS, 103, p. 161 (1995)). IFN-.gamma. exerts its effects in part by modulating the expression of genes through the STAT and IRF pathways Schindler, C. and Darnell, J. E. Ann. Rev. Biochem., 64, p. 621 (1995); Taniguchi, T. J. Cancer Res. Clin. Oncol., 121, p. 516 (1995)).
Mice lacking IFN-.gamma. or its receptor have multiple defects in immune cell function and are resistant to endotoxic shock (Huang, S. et al., Science, 259, p.1742 (1993); Dalton, D. et al., Science, 259, p. 1739 (1993); Car, B. D. et al., J. Exp. Med., 179, p.1437 (1994)). Along with IL-12, IGIF appears to be a potent inducer of IFN-y.gamma. production by T cells (Okamura, H. et al., Infection and Immunity, 63, p.3966 (1995); H. Okamura et al., Nature, 378, p.88 (1995); Ushio, S. et al., J. Immunol., 156, p.4274 (1996)).
IFN-.gamma. has been shown to contribute to the pathology associated with a variety of inflammatory, infectious and autoimmune disorders and diseases. Thus, compounds capable of decreasing IFN-.gamma. production would be useful to ameliorate the effects of IFN-.gamma. related pathologies.
IGIF is synthesized as a precursor protein, called "pro-IGIF". Recently, ICE and other members of the ICE/CED-3 family have been linked to the conversion of pro-IGIF to IGIF or to IFN-.gamma. production in vivo (see PCT patent application WO 97/22619, which is incorporated herein by reference).
Accordingly, compositions and methods capable of regulating the conversion of pro-IGIF to IGIF would be useful for decreasing IGIF and IFN-.gamma. production in vivo, and thus for ameliorating the detrimental effects of these proteins which contribute to human disorders and diseases.
ICE inhibitors represent a class of compounds useful for the control of inflammation or apoptosis or both. Peptide and peptidyl inhibitors of ICE have been described. PCT patent applications WO 91/15577; WO 93/05071; WO 93/09135; WO 93/14777 and WO 93/16710; and European patent application 0 547 699. Such peptidyl inhibitors of ICE have been observed to block the production of mature IL-1.beta. in a mouse model of inflammation (vide infra) and to suppress growth of leukemia cells in vitro (Estrov et al., Blood 84, 380a (1994)). However, due to their peptidic nature, such inhibitors are typically characterized by undesirable pharmacological properties, such as poor cellular penetration and cellular activity, poor oral absorption, poor stability and rapid metabolism. Plattner, J. J. and D. W. Norbeck, in Drug Discovery Technologies, Clark, C. R. and Moos, W. H. Eds. (Ellis Horwood, Chichester, England, 1990), pp.92-126. This has hampered their development into effective drugs.
Non-peptidyl compounds have also been reported to inhibit ICE in vitro. PCT patent application WO 95/26958; U.S. Pat. No. 5,552,400; Dolle et al., J. Med. Chem., 39, pp. 2438-2440 (1996). However, it is not clear whether these compounds have the appropriate pharmacological profiles to be therapeutically useful.
Additionally, current methods for the preparation of such compounds are not advantageous. These methods use tributyltin hydride, a toxic, moisture-sensitive reagent. Thus, these methods are inconvenient to carry out, pose a health risk and create toxic-waste disposal problems. Furthermore, it is difficult to purify compounds prepared by these methods. A preferred method for preparing compounds, such as the ICE inhibitors of this invention, has been described in PCT patent application WO 97/22619, which is incorporated herein by reference.
Accordingly, the need exists for compounds that can effectively inhibit the action of ICE in vivo, for use as agents for preventing and treating chronic and acute forms of IL-1-mediated diseases, apoptosis-, IGIF-, or IFN-.gamma.-mediated diseases, as well as inflammatory, autoimmune, destructive bone, proliferative, infectious, or degenerative diseases. The need also exists for methods of preparing such compounds.